A typical flow cytometer includes an exciter, an interrogation zone, and a detector. The detector functions to collect signals from objects, ranging from small to large, in the interrogation zone. The typical detector has a limited collection range, and is typically provided with a gain level. To collect signals from small objects, the gain level is increased. With an increased gain level, however, the signals from large objects are too bright to be collected. Accordingly, to collect signals from large objects, the gain level is decreased. With a decreased gain level, however, the signals from small objects are too faint to be collected. Thus a fundamental problem with flow cytometry is the selection of gain levels for the amplifiers operating on the detector signal.
In quantitative terms, the collection range for a typical flow cytometer with a photomultiplier tube is three to four decades, where a decade is defined as a logarithmic measurement interval consisting of a multiplication or division by a power of ten. In flow cytometry, the signal range of the objects may span five or more decades across experiments, i.e. a range of 1-100,000 units or more between data points. As such, the collection range of a typical flow cytometer is smaller than the signal range of the objects. For this reason, users are typically required to pre-set the gain levels of the detector by setting one or more voltages for the photomultiplier tubes and/or amplifier gains such that they correspond to an anticipated range of data points for the objects.
The requirement of this set-up step, which must occur before the collection of any data, has several disadvantages. One disadvantage, the user must expend valuable time and energy in anticipating the appropriate range for collection and data processing. A second disadvantage, any pre-selection of the detector gain necessarily includes the potential loss of valuable data because the user incorrectly anticipated the actual signal range and a portion or more of the input signals are outside the user-set collection range and are not collected.
Another disadvantage is the difficulty related to the concept of spectral spillover. Most fluorochromes used in flow cytometer analyses have broad emission spectra and therefore produce detectable signals in multiple channels. Typically, a fluorochrome will have the majority of its signal detected by only one detector, which is defined (by use of an appropriate light filter) as the primary detector. Fluorescence detected by detectors, other than that defined as the primary, is termed “spillover”. (Spillover is actual fluorescence, not an artifact, as the term would imply.) Spectral compensation refers to a mathematical algorithm that allows the operator to subtract spillover fluorescence signals from any channel. Applying spectral compensation to a data set changes the fluorescence channel number assigned to events in the “spillover” (non-primary) detector by subtracting a predetermined percentage of the original channel number. The amount of spillover for any given fluorochrome into neighboring channels is dependent on a number of factors, including (but not limited to) the fluorochrome, the laser excitation wavelength and power, the optic configuration for light collection, the light filters used, the sensitivity of the detectors, and the amount of amplification (voltage and/or gain) applied to the detectors. Thus, the flow cytometer setup procedure is a great disadvantage since variables associated with the detector factor into the compensation of spectral spillover.
Accordingly, there is a need in the art to create a new and improved detection system for a flow cytometer that avoids or minimizes these disadvantages. The present invention provides such new and improved detection system for a flow cytometer.